Strategies for cellular therapy in vascular medicine are being developed to treat refractory claudication, but are limited by the resident population of vascular endothelial cells in adults that is competent to I respond to an available level of angiogenic growth factors. Umbilical cord blood derived endothelial precursor cells (UCB derived EPCs) have advantages of greater lifespan and reparative proliferation, relative to existing models of therapeutic angiogenesis derived from patient peripheral blood or bone marrow, and do not incur the ethical controversy of use of embryonic stem cells. This application outlines a highly interactive group of investigators focused on EPCs with laboratory studies directed to a further understanding of EPC differentiation and to optimize ex vivo expansion conditions. Since little is known of the cytokine and gene regulation in EPC ontogeny, our studies in a SCID.NOD murine study model of hind-limb ischemia will be conducted with concomitant in vitro cellular and gene array analyses in order to gain further understanding of EPC development from early AC133+ stem cells, as well as gene regulation of EPC differentiation. We propose to isolate and expand unselected and AC133 selected human UCB into EPCs. Optimum media and growth factor combinations will be determined and expanded cells will be transplanted into a murine model of hindlimb ischemia to determine the effects of cell infusion on angiogenesis. Blood flow will be measured with laser Doppler perfusion imaging, and capillary density in the affected and control limb compared. HLA analysis will be conducted to identify and compare 3referential grafting by specific cellular subsets. Current studies incorporating infusion of culture expanded EPC point to cells derived from immature hemangioblasts as well as mature endothelial cells derived from vascular intima as injury response. It is unclear the relative contributions of these cell populations to home and effect angiogenesis. Since the number and function of EPC decline with advancing age, UCB may be considered as a robust source of rapidly proliferating EPCs for therapeutic angiogenesis in the treatment of ischemic vascular disease.